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J Struct Biol. 2016 Jun;194(3):303-10. doi: 10.1016/j.jsb.2016.03.006. Epub 2016 Mar 8.

Structure of γ-tubulin small complex based on a cryo-EM map, chemical cross-links, and a remotely related structure.

Author information

1
Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, California Institute for Quantitative Biosciences, University of California at San Francisco, San Francisco, CA, USA. Electronic address: cgreen@salilab.org.
2
Department of Biochemistry, University of Washington, Seattle, WA, USA.
3
Department of Genome Sciences, University of Washington, Seattle, WA, USA.
4
Department of Genome Sciences, University of Washington, Seattle, WA, USA. Electronic address: tdavis@uw.edu.
5
Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA, USA; Howard Hughes Medical Institute, University of California at San Francisco, San Francisco, CA, USA. Electronic address: agard@msg.ucsf.edu.
6
Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, California Institute for Quantitative Biosciences, University of California at San Francisco, San Francisco, CA, USA. Electronic address: sali@salilab.org.

Abstract

Modeling protein complex structures based on distantly related homologues can be challenging due to poor sequence and structure conservation. Therefore, utilizing even low-resolution experimental data can significantly increase model precision and accuracy. Here, we present models of the two key functional states of the yeast γ-tubulin small complex (γTuSC): one for the low-activity "open" state and another for the higher-activity "closed" state. Both models were computed based on remotely related template structures and cryo-EM density maps at 6.9Å and 8.0Å resolution, respectively. For each state, extensive sampling of alignments and conformations was guided by the fit to the corresponding cryo-EM density map. The resulting good-scoring models formed a tightly clustered ensemble of conformations in most regions. We found significant structural differences between the two states, primarily in the γ-tubulin subunit regions where the microtubule binds. We also report a set of chemical cross-links that were found to be consistent with equilibrium between the open and closed states. The protocols developed here have been incorporated into our open-source Integrative Modeling Platform (IMP) software package (http://integrativemodeling.org), and can therefore be applied to many other systems.

KEYWORDS:

Allosteric regulation; Comparative models; Computational structure prediction; Integrative modeling; Microtubule nucleation

PMID:
26968363
PMCID:
PMC4866596
DOI:
10.1016/j.jsb.2016.03.006
[Indexed for MEDLINE]
Free PMC Article

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